脂质体介导NF-κB圈套寡核苷酸体外转染Kupffer细胞的实验研究  

In vitro lipofectamine mediated NF-κB decoy oligodeoxynucleotides transfection of Kupffer cells

作　　者：彭勇[1] 李敬东[1] 肖江卫[1] 李旭红[2] 甘霖[2] 龚建平[2] 

机构地区：[1]川北医学院附属医院普外三科,南充637000 [2]重庆医科大学附属第二医院肝胆外科

出　　处：《中华普通外科杂志》2010年第6期472-475,共4页Chinese Journal of General Surgery

基　　金：国家自然科学基金资助项目（30300337,30700773）

摘　　要：目的 研究核因子κB（NF-κB）圈套寡核苷酸（decoy oligodeoxyribonucleotide,ODN）体外借助阳离子脂质体转染大鼠Kupffer细胞（KCs）的效率,并观察其对KCs活化的抑制作用.方法 24只Wistar大鼠分为3组,每组8只.（1）对照组：分离培养正常大鼠KCs （2）LPs组：KCs培养液中加入终浓度为1 ms/L的LPS作为刺激因素 （3）NF-κB圈套寡核苷酸组：先用阳离子脂质体（lipofectamine）将人工合成的NF-κB圈套寡核苷酸转染KCs（4μg/1×10^5个KCs）,观察转染效果,再用1 mg/L LPS刺激,观察其对KCs活化的抑制作用,包括KCs的吞噬功能、NF-κB易位,以及膜表面分子CD4O mRNA的表达.结果 在LPS刺激下,KCs呈高度活化状态,吞噬功能增强,NF-κB向细胞核移位,细胞膜表面共刺激分子CD40 mRNA呈高表达状态,与对照组相比差异有统计学意义（t=4.01,P〈0.01）.在阳离子脂质体介导下,人工合成的NF-κB圈套ODN可以高效转染KCs,有效地抑制NF-κB活化,降低其下游基因的表达,与LPS组相比差异有统计学意义（t=4.89,P〈0.01）.结论 在脂质体介导下,NF-κB圈套寡核苷酸可以高效转染KCs,并有效抑制KCs的活化.Objective To study the transfection effects of nuclear factor-KappaB（NF-κB）decoy oligodeoxynucleotides（ODN） to Kupffer cells （KCs） mediated by lipofectamine,and investigate it＇s suppression effects on KCs activation. Methods Twenty-four Wistar rats were divided into three groups （n=8）.（1）Control group,in which the normal KCs were isolated.（2）LPS group,in which 1 ms/L LPs was added to the culture system.（3）NF-κB decoy ODN group,in which KCs were transduced with NF-κB decoy ODN （4μg×10^5KCs）prior to LPS stimulation.The transfection efficiency Was assayed,and the phagocytosis function,NF-κB（P65） translocation,CD40 mRNA expression of KCs were also detected respectively. Results Kupffer cells were obviously activated after LPS stimulation.the phagocytosis function was reinforced.the activity of NF-κB transloeated from cytoplasm into nucleus was obviosly increaced.The co-stimulatory molecules expression（CD40 mRNA）significantly increased compared with control group（t=4.01,P〈0.01）.NF-κB decoy oligodeoxynucleotides can efficiently transfected into KCs mediated by lipofectamine,which can obviously suppress KCs activation,and downregulate the expression of downstream gene（compared with LPS group,t=4.89,P〈0.01）. Condusion NF-κB decoy ODN can efficiently transfect into KCs and inhibit it＇s activation.

关 键 词：枯否细胞 NF-ΚB 圈套寡核苷酸 

分 类 号：R657.3[医药卫生—外科学]